Primer directed nucleic acid amplification including the addition of specific template DNA inactivating enzyme

Abstract

Methods for the enzymatic reduction and/or elimination of contaminant DNA from enzymatic amplification procedures are disclosed. These methods include the use of restriction endonucleases, DNAases and exonucleases which act upon contaminant DNA in polymerase chain reaction mixtures such that contaminant DNA is not amplified.

I claim: 
     
       1. A method for the inactivation of contaminant PCR product DNA templates in an amplification reaction mixture, said mixture having at least one contaminant PCR product DNA template, at least one target DNA template, complementary primers to said target DNA template, a heat resistant DNA polymerase and a supply of dNTPs under conditions suitable for maintaining a polymerase chain reaction, which comprises: a) contacting said mixture With at least one enzyme selected from the group consisting of exonucleases and other enzymes with exonucleolytic properties;   b) incubating said mixture while being contacted with said at least one enzyme at a temperature sufficient and for a time sufficient to permit inactivation of said contaminant PCR product DNA template;   c) incubating the product of step b) at a temperature sufficient and for a time sufficient to permit inactivation of said exonucleases; and   d) cycling the product of step c) at temperatures and for times sufficient to permit amplification of the target DNA template.   
     
     
       2. The method of claim 1 wherein the enzyme is T7 exonuclease (gene 6). 
     
     
       3. The method of claim 1 wherein the enzyme is exonuclease III. 
     
     
       4. The method of claim 1 wherein the enzyme is Bal 31 nuclease. 
     
     
       5. The method of claim 1 wherein the heat resistant DNA polymerase is Taq DNA polymerase. 
     
     
       6. The method of claim 1 wherein said exonuclease is T4 DNA polymerase.